Mutation and dysregulation of the cardiac ryanodine receptor (RyR2) or sarcoplasmic reticulum (SR) calcium release channel contributes directly to catecholaminergic polymorphic ventricular tachycardia (CPVT) and heart failure (HF) in humans. Affected RyRs are more active than normal, leaking Ca from the SR during diastole causing arrhythmias and dysfunction. FKBP12 and FKBP12.6 are small proteins that bind tightly with RyR1 and RyR2, respectively. FKBP is thought to stabilize the resting RyR. Moreover, dysregulation of the RyR-FKBP association due to RyR hyperphosphorylation has been proposed to explain contractile dysfunction and arrhythmogenesis in HF, and exercise-induced sudden cardiac death. Thus, the FKBP/RyR interface is a promising new therapeutic target for arrhythmias and HF. However, this field is controversial and solid fundamental studies are needed to move this field forward. Indeed, there is incomplete molecular understanding of the FKBP/RyR interaction, (both structurally and functionally), and limited data about FKBP12/12.6 binding and effects in the myocyte environment. Here we will define the molecular architecture of the FKBP/RyR interaction, and test its functional relevance in cardiac myocytes and isolated SR and RyRs (using fluorescent- tagged FKBP and fluorescence resonance energy transfer (FRET). Four Specific Aims will: 1) identify key FKBP12 & 12.6 residues at the interface with RyR2 (& RyR1), using cysteine- scanning and site-directed labeling of FKBP, 2) use functionally silent selected fluorescent FKBPs from aim 1 to assess FKBP-RyR affinity, on- & off-rates in myocytes with simultaneous functional RyR2 readout as Ca sparks (under key different conditions), 3) measure physical distances and orientation between critical sites in the RyR2 complex in situ using FRET between FKBP and calmodulin, RyR2 itself and other complex components, and 4) measure RyR FKBP12/12.6-CaM conformational changes induced by physiological RyR modulation using novel optical probes. This is a highly collaborative project between two groups with shared interests and complementary expertise spanning fluorescence spectroscopy, biochemistry, molecular biology and confocal imaging. This will greatly enhance our understanding of RyR2 function in cardiac myocytes and how (and where) FKBP modulates RyR2. PUBLIC HEALTH RELEVANCE: The ryanodine receptor (RyR) is the intracellular calcium release channel that is critical in both driving the normal cardiac contraction and also life-threatening arrhythmias. RyR dysfunction is implicated in human heart failure and arrhythmias, and the small regulatory protein FKBP has been suggested to be at the heart of the problem, but how that may occur is controversial. We will conduct fundamental quantitative molecular fluorescence studies that will clarify both structurally and functionally how FKBP binds to and regulates RyR function in the heart.